This laboratory has used gene expression analysis involving in situ hybridization and DNA microarray technology to identify genes that function in embryonic development. The initial dorsal-ventral axis of the zebrafish embryo is established by transcriptional control exerted by the Wnt pathway effector molecule beta-catenin, which becomes localized in the nucleus of one side of the early embryo. This side will form the organizer at the early gastrula stage. One of the first consequences of -catenin function is activation of the homeobox gene encoding the transcriptional repressor Bozozok/Dharma/Nieuwkoid (hereafter called Boz). Boz functions by repressing ventral genes such as vent, vox, ved and bmp. We found that the maternally inherited E3 ubiquitin ligase Lnx-2b (previously known as Lnx-like) can bind and ubiquitilate Boz, targeting it for proteasomal degradation. If Lnx-2b is depleted from the embryo, the embryo is dorsalized because of expansion of the domain in which Boz is active, consequently increasing the size of the organizer. The presence of Lnx-2b balances the organizer and ventral domains in the embryo, assuring appropriate axis formation and normal development. These studies point to the modulation of protein stability as a fundamental mechanism in the regulation of early embryogenesis in the zebrafish. An extensive DNA microarray analysis was carried out to characterize the RNA populations in the zebrafish pineal gland, as described in a previous Annual Report. Among the genes found to be enriched specifically in the pineal gland we identified a homolog of the unc119 gene initially described in Caenorhabditis elegans. Two unc119 paralogs have been described in mammals, with expression in the brain and retina. In zebrafish, two unc119 genes have been identified previously in other laboratories. Thus we named the gene whose expression we found to be highly enriched in the pineal gland as unc119c. We have shown that the Unc119c protein can interact physically with a small GTPase named Arl3l2, suggesting a possible function in intracellular protein trafficking. The role of Unc119c in the pineal gland is being investigated. A focus of interest in this laboratory has been the study of genes that are involved in the formation of the neural crest and of its derivatives such as the pharyngeal arches. Two studies were conducted in this area in the recent period. In the first we analyzed the BTB-domain containing protein Kctd15 that is first expressed in the embryo in the neural plate border and subsequently in pharyngeal arches and other regions. Overexpression of Kctd15 strongly inhibits neural crest specification, while attenuation of Kctd15 expression leads to expansion of the neural crest precursor domain. We found that Kctd15 inhibits the output of the canonical Wnt signaling pathway upstream of or at the level of -catenin;Wnt signaling is known to be crucial for induction of the neural crest and for distinguishing neural crest precursors from precursors of anterior placodes that also arise at the neural plate border. We propose that Kctd15 is involved in delineating the separate placode and neural crest domains during embryogenesis. These studies were carried out both in zebrafish and in Xenopus, using zebrafish embryos primarily for whole-embryo analysis and Xenopus for explant analysis. A second project concerned the analysis of differentiation of the craniofacial skeleton that prominently involves neural crest-derived cells. The zebrafish Barx1 gene has been isolated and shown to be involved in the regulation of chondrogenesis during pharyngeal arch development. Barx1 is expressed in the cranial neural crest, the pharyngeal arches, the anterior aspect of the pectoral fin buds, and the gut wall. By 2.5 days post fertilization, embryos in which Barx1 expression was inhibited show developmental delay exemplified by poor facial outgrowth and micrognathia. Histological analysis and labeling of cell membranes revealed reductions in differentiation and chondrocyte condensation within the arches. Affected larvae stained with Alcian blue exhibit small and dysmorphic arch cartilage elements, and expression of chondrogenic markers is perturbed. The expression of Barx1 is controlled by bone morphogenetic protein (Bmp) as seen in bead implantation experiments. These results suggest a role for Barx1 at early stages of chondrogenesis within the pharyngeal arches during zebrafish development. It is possible that these observations will become relevant to the study of malformation in human craniofacial development. The role of FGF signaling in embryogenesis has been a long-standing interest of our laboratory. In a recent collaborative study we found that three Ets transcription factors in the Pea3 family are the major effectors of FGF signaling in the zebrafish embryo. Simultaneous reduction of expression of all three Pea3 factors largely mimicked the effect of inhibiting FGF signaling in the embryo. In particular, the development of the heart and the establishment of left/right asymmetry were affected in embryos deficient in the three Pea3 family transcription factors. Differentiation of the posterior region of the body requires the cooperation of signaling pathways such as the Fgf, Bmp, Wnt, and retinoic acid pathways together with the action of several transcription factors that are both targets and modulators of these signaling pathways. The nature of these functional interactions has been explored in a collaborative study. In this work we analyzed the role of the homeodomain transcription factor Eve1, a member of the Evx family, in axis establishment and neural induction in the zebrafish embryo. Eve1 is required for tail formation, in particular for the differentiation of posterior neural tissue in the embryo. Eve1 accomplishes its role in two ways first, Eve1 can induce aldh1a2, a gene that encodes a biosynthetic enzyme that is in the pathway of retinoic acid production. Second, Eve1 attenuates Bmp expression, which is a necessary condition for the induction of neural tissue. Thus Eve1 was shown to be a key factor in the formation of posterior neural derivatives.